Laser printing method and device for implementing said method

ABSTRACT

A method for printing uses at least one ink. The method includes: a step of focusing a laser beam so as to generate a cavity in an ink film; a step of forming at least one ink droplet from a free surface of the ink film; and a step of depositing the droplet onto a depositing surface of a receiving substrate positioned at a given distance from the film. The laser beam is oriented in the direction opposite the gravitational force. The free surface of the film is oriented upwards towards the depositing surface placed above the ink film.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a National Phase Entry of International PatentApplication No. PCT/FR2015/053569, filed on Dec. 17, 2015, which claimspriority to French Patent Application Serial No. 1462568, filed on Dec.17, 2014, both of which are incorporated by reference herein.

BACKGROUND AND SUMMARY

The present invention relates to a method for laser printing and to adevice for the implementation thereof.

Ink printing is used in many fields to reproduce complex designs. Theprinting of elements can thus notably be implemented in fields as variedas biology, electronics, materials or clockmaking. The problems met inthese fields are similar and relate in particular to needs for makingelements combinations on a very small scale. A reproduction of patternswhich consists in depositing material at specific locations can bechemically or physically performed by using masks or resorting to a stepof selective ablation.

To overcome the disadvantages of these methods (risk of contamination,complex implementation, difficult combination of the deposition ofseveral elements), ink printing methods have been developed. These havethe advantage of being adapted to very freely generate element patternsusing computer-aided design tools which they are associated with.

In the field of biology, depending on the works, such printing methodsare called bio-printing, micro-printing of biological elements or simplybio-printing. According to these methods, the biological tissue isobtained by printing droplets of bio-inks. In order to reach somevolume, the droplets are arranged in layers which are stacked on eachother.

In a first embodiment, ink is stored in a tank and goes through nozzlesor capillaries in order to form droplets that are transferred onto asubstrate. This first alternative solution, so-called a nozzle printingincludes bio-extrusion, ink-jet printing or micro-valves printing.Bio-extrusion makes it possible to obtain a significant cell density ofthe order of 100 million cells per milliliter and a resolution of onemillimeter. Micro-valves printing makes it possible to obtain a lessercell density of the order of several million cells per milliliter and abetter resolution of the order of 100 μm. Ink-jet printing makes itpossible to obtain a cell density identical with that of micro-valvesprinting, of less than 10 million cells per milliliter and a betterresolution of the order of 10 μm.

In the case of bio-extrusion, the cells are deposited from a firstnozzle and a hydrogel is simultaneously deposited from a second nozzle.As an alternative solution, the cells and the hydrogel are mixed in atank prior to the extrusion. In the two other cases, ink is an aqueousmedium containing the cells. According to the alternative solutions,bio-extrusion makes it possible to deposit the ink continuously asfilaments or discontinuously as droplets.

According to such nozzle printing modes, as the printing resolution islinked in particular to the nozzle section, only bio-inks with givenrheological characteristics can be used for high resolutions. Bio-inkswith high cell density can thus be printed with difficulty, with a highresolution since such printing technique induces, upon the passage ofink through the nozzle, high shear stresses liable to damage the cells.Besides, with this type of ink, the risk of nozzles being blocked by thecells is important mainly because of the sedimentation of cells insidethe tanks.

A method for printing biological elements by laser has been developed inorder to be able to use a wide range of bio-inks and achieve highresolution. This printing method, called laser bio-printing, is alsoknown as “Laser-Assisted Bio-printing” (LAB). The invention specificallyrelates to this type of printing methods. For comparison, bio-laserprinting makes it possible to print inks with a high cell density of theorder of 100 million cells per milliliter with a resolution of 10 μm.Similarly, laser printing has also been developed in other fields inorder to improve resolution and expand the range of usable inks.

Compared to the nozzle printing techniques, laser printing providesgreater flexibility in use (ability to print on soft, uneven surfaces .. . ), reduces shear stress, and limits the risk of settling. Accordingto another advantage, it is possible to print from a small volume of inkof the order of a few microliters, which is interesting for deposits ofexpensive materials. Eventually, it is possible to use the printingsystem to view and select the drop region as described in WO2011/107599.

As illustrated in FIG. 1, a device for laser printing biologicalelements which is based on the so-called “Laser-Induced ForwardTransfer” (LIFT) technique, comprises a pulsed laser source 10 emittinga laser beam 12, a system 14 for focusing and orienting the laser beam12, a donor substrate 16 which comprises at least one bio-ink 18 and areceiving substrate 20 so positioned as to receive droplets 22 emittedfrom the donor substrate 16. According to this printing technique, thelaser beam is pulsed and a droplet is generated on each pulse.

Bio-ink 18 comprises a matrix, for example an aqueous medium, whereinelements are present, for example, cells, to be deposited onto thereceiving substrate 20. The donor substrate 16 comprises a blade 24transparent to the wavelength of the laser beam 12 which is coated withan absorbent layer 26 whereon bio-ink 18 is affixed as a film. Theabsorbent layer 26 makes it possible to convert light energy intokinetic energy. The laser beam 12 thus produces a punctual heating atthe absorbent layer 26 that generates, by vaporization, a gas bubble 28which, by expansion, causes the ejection of a droplet 30 of bio-ink.

According to a known arrangement, the laser beam 12 impacts the donorsubstrate 16 by being oriented in an approximately vertical directionand in an upward direction, or in the same direction as thegravitational force G. Bio-ink 18 is thus placed under the blade 24 soas to be oriented downwards, towards the receiving substrate 20 which isplaced under the donor substrate 16. Given this arrangement, bio-ink 18is in the form of a film with a thickness E lower than a given thresholdto be held on the blade. This threshold varies in particular accordingto the surface tension, viscosity and density of bio-ink.

The formation of droplets 30 from the ink film depends on manyparameters which relate in particular to the laser beam 12 (wavelength,energy, pulse duration, . . . ), the nature of the bio-ink 18 (surfacetension, viscosity, . . . ), to external conditions (temperature,humidity, . . . ). The formation of droplets 30 also depends on thethickness E of the bio-ink film. The droplets will not form if thethickness E of the bio-ink film is not included in a thickness rangedefined by a lower bound and an upper bound. If the thickness E has avalue above the upper limit, no droplet will form because the expansionof the gas bubble 28 is too weak to reach the free surface of the film.If the thickness E has a value under the lower limit, the gas bubblewill burst 28 at the free surface causing the uncontrolled projection ofa plurality of micro-droplets toward the receiving substrate.

Therefore, the film thickness E has to be substantially constant overthe entire surface of the donor substrate 16 to obtain reproducibilityof the droplet formation whatever the region of the donor substrate 16affected by the laser beam 12. Now, as illustrated in FIG. 1, thisthickness E is not constant. This reproducibility issue is not limitedto the case of bio-inks. It is present whatever the field of use, duringthe laser printing of an ink film.

To remedy this problem, a publication entitled “Microdroplet depositionthrough a film-free laser forward technique” published on Oct. 1, 2011on the site www.elsevier.com provides for a device as described in FIG.2. As before, this device comprises a laser source 32 emitting a laserbeam 34, a system 36 for focusing and orienting the laser beam 34, adonor substrate 38 which contains at least one bio-ink 40 and areceiving substrate 42 positioned to receive droplets 44 emitted fromthe donor substrate 38. According to this publication, the donorsubstrate 38 includes a tank 46 with no upper wall so that the freesurface 48 of the bio-ink 40 contained in the tank faces the receivingsubstrate 42. To obtain a regular, substantially planar, free surface 48bio-ink is not a thin film but a volume having a depth of the order of 3mm. Thus, the tank bottom has no influence on the shape of the freesurface 48 of the bio-ink and the side walls of the tank have a limitedeffect at the periphery of the free surface 48 because of the surfacetension.

Given the depth of the volume of bio-ink, the free surface 48 isnecessarily directed upwards to stay in the tank and the receivingsubstrate 42 is positioned above the bio-ink 40. According to thisdocument, to obtain the ejection of a droplet, the laser beam 34 isfocused just below the free surface 48 and has a depth of the order of40 to 80 μm. Thus, the droplets emitted from the free surface 48 areprojected toward the receiving substrate 42 in a direction of movementopposite the direction of the gravitational force G.

Although the solution proposed by this publication makes it possible toobtain a flat free surface 48 for the ink, it is not necessarily adaptedto inks in the form of suspensions, such as bio-inks. As a matter offact, as indicated above, such bio-inks contain elements to be printed,such as, for instance cells, embedded in a matrix, which tend to settledown on the tank bottom. As the concentration in elements to be printedis low near the free surface, the printed droplets have de facto lowconcentrations in cells, which is generally detrimental to the printedbiological tissue. Besides, according to this method, the number ofcells and the concentration in deposited cells can hardly be controlled.

Such settlement issue is not limited to bio-inks. Thus, it is foundduring the laser printing of inks as suspensions, such as suspensions ofparticles or nanoparticles in a liquid matrix, whatever the field ofapplication of such inks. According to another disadvantage of themethod of the publication, the ink must be able to absorb the laserbeam, which could limit the range of inks that can be printed using thistechnique.

The present invention therefore aims to remedy the drawbacks of theprior art by providing a printing method which makes it possible toprint a wide range of elements with great accuracy. In particular, thismethod makes it possible to print a wide range of biological elements,specifically so as to obtain complex biological tissues. For thispurpose, the invention relates to a method for printing with at leastone ink, with said method comprising a step of focusing a laser beam soas to generate a cavity in an ink film, a step of forming at least oneink droplet from a free surface of the ink film and a step of depositingsaid droplet onto a depositing surface of a receiving substratepositioned at a given distance from the film, characterized in that thelaser beam is oriented in the direction opposite the gravitationalforce, with the free surface of the film being oriented upwards towardsthe depositing surface placed over the ink film.

This configuration makes it possible, in particular, to obtain asubstantially constant thickness E for the ink film, while limiting theoccurrence of settling phenomena. Besides, it makes it possible to use awide range of inks. The ink printed using the method according to theinvention can be any liquid ink and can be a solution or a suspension.

Bio-inks, the inks used in electronics or watchmaking can be cited,among the usable inks. According to one application, the ink is abio-ink.

According to another characteristic, the film has a thickness of lessthan 500 μm and/or has a dimension of the free surface of the film onfilm thickness ratio greater than or equal to 10. The distanceseparating the ink film and the depositing surface and/or the beam laserenergy are preferably so adjusted that the kinetic energy of the dropletis almost equal to zero when the droplet contacts the depositingsurface. Such characteristic limits the risks of damaging the elements(cells or other elements) contained in the droplet. According to oneembodiment, the distance separating the ink film and the depositingsurface is equal to 1 to 2 mm and the beam laser energy is so adjustedthat the kinetic energy of the droplet is almost equal to zero when thedroplet contacts the depositing surface.

According to another characteristic, the printing method comprises apreliminary phase of calibration of the laser beam energy. Thiscalibration phase comprises a step of measuring an included angle of adeformation of the free surface of the ink film at a set time after theimpact of the laser beam and a step of adjusting the laser beam energyas a function of the measured value of the included angle.

The laser beam energy is so adjusted that the included angle is lessthan or equal to 105°. In this case, the laser beam energy is sufficientto cause the formation of a droplet. The energy of the laser beam isadvantageously so adjusted that the included angle is greater than orequal to a second threshold to obtain a kinetic energy almost equal tozero at the time when the formed droplet reaches the depositing surface.

For an ink, preferably a biological ink, film with a thickness of theorder of 40 and 50 μm, the time for measuring the included angle ispreferably of the order of 4 to 5 μs from the impact of the laser beam.Advantageously, the ink film has a thickness greater than 20 μm. For anink which is in suspension with a high concentration in elements to beprinted, the ink film preferably has a thickness ranging from 40 to 60μm. Advantageously, in order to improve the accuracy of the depositionof the elements to be printed, the ink film 74 has a thickness E between1.5D and 2D, with D being the diameter of the elements to be printedwhich have an approximately spherical shape or the diameter of a spherein which at least one element to be printed is inscribed.

The invention also relates to a printing device for implementing theprinting method of the invention. It comprises:

-   -   at least a pulsed laser source so configured as to emit a laser        beam,    -   an optical system for focusing and orienting said laser beam,    -   at least one donor substrate which at least an ink film is        attached to with a free surface, and    -   at least one receiving substrate comprising a depositing        surface.        The printing device is characterized in that the laser beam is        oriented in the direction opposite the gravitational force and        in that the free surface of the film is oriented upwards towards        the depositing surface placed above the ink film.

BRIEF DESCRIPTION OF THE DRAWINGS

Other characteristics and advantages will appear from the followingdescription of the invention, and such description is given by way ofexample only, with reference to the appended drawings in which:

FIG. 1 is a schematic representation of a laser printing device whichillustrates an alternative solution of the prior art;

FIG. 2 is a schematic representation of a laser printing device whichillustrates another alternative solution of the invention;

FIG. 3 is a schematic representation of a laser printing device whichillustrates the invention;

FIGS. 4A to 4D are side views illustrating the formation or not of adroplet according to different rates;

FIGS. 5A to 5D are diagrams illustrating a droplet at different times ofits formation, with the last FIG. 5D illustrating the time when adroplet reaches a receiving substrate;

FIG. 6 is a section of a donor substrate illustrating the relationshipbetween the size of the elements to be printed and the thickness of abio-ink film;

FIGS. 7A and 7B are side views illustrating the formation of aprotrusion on the free surface of a bio-ink film prior to the formationof a droplet, produced at the same time but with different energies forthe laser beam;

FIG. 8 is a schematic representation of a printing device according toone embodiment of the invention which combines at least one laser-typeprint head and at least one ink-jet-type print head;

FIG. 9 is a perspective view of a printing device according to oneembodiment of the invention which combines a laser-type print head andseveral ink-jet-type print heads;

FIG. 10 is a perspective view of a part of the printing device of FIG. 9when printing with an ink-jet-type print head;

FIG. 11 is a sectional view of a part of the printing device of FIG. 9when printing with an laser-type print head;

FIG. 12 is a perspective view of a 3-dimensional representation of aportion of a biological tissue which is to be replicated;

FIG. 13 is a perspective view of a slice of the representation of FIG.12; and

FIG. 14 is a top view of the slice of FIG. 13 illustrating thepositioning of the bio-inks droplets.

DETAILED DESCRIPTION

FIG. 3 shows a printing device 50 for producing at least one biologicaltissue through a layer by layer assembling according to a predefinedarrangement, of various components such as an extracellular matrix andvarious morphogens. Thus, the printing device 50 makes it possible todeposit, layer by layer, droplets 52 of at least one bio-ink 54 onto adepositing surface 56 which corresponds to the surface of a receivingsubstrate 58 for the first layer or for the last layer deposited on saidreceiving substrate 58 for the following layers. In order to simplifythe representation, the depositing surface 56 corresponds to the surfaceof the receiving substrate 58 in FIG. 3.

According to one embodiment in FIG. 6, the bio-ink 54 comprises a matrix60, for example an aqueous medium, wherein elements 62, for example,cells or cell aggregates, to be printed onto the depositing surface 56can be found. As the case may be, a bio-ink 54 comprises, in the matrix60, only one kind of elements to be printed 62 or several kinds ofelements to be printed 62. In an alternative solution, the bio-ink 54may comprise one component only.

Bio-ink means, for the present patent application, a biological materialor bio-material. For example, the bio-ink only comprises anextracellular matrix (for instance collagen), an extracellular matrixand elements such as cells or cell aggregates, an aqueous mediumcontaining elements such as cells or cell aggregates. The bio-ink 54 isnot further described because it can have different types and differentrheological characteristics from one ink to another.

This printing device comprises a laser source 64 so configured as toemit a laser beam 66 which is characterized specifically by itswavelength, its frequency, its energy, its diameter, its pulse duration.Preferably, the laser source 64 can be so configured as to adjust atleast one characteristic of the laser beam, in particular the energythereof.

In order to form droplets separated from each other, the laser source 64is a pulsed source. To give an order of magnitude, 10,000 droplets canbe ejected per second. For example, the laser source 64 is a lasersource with a wavelength of 1,064 nm.

In addition to the laser source, the printing device 50 includes anoptical system 68 which enables the adjustment of the focus along a Zaxis perpendicular to the depositing surface 56. The optical system 68advantageously includes a lens which makes it possible to focus thelaser beam 66 onto an impacted area. The optical system 68 preferablyincludes a mirror to change the position of the impacted area. Theoptical system 68 thus makes it possible to change the area impacted bythe laser beam in an impact plane referenced Pi in FIG. 3. The lasersource 64 and the optical system 68 are not further described either,since they are known to the persons skilled in the art and may beidentical with those of the prior art.

The printing device 50 also comprises at least one donor substrate 70which comprises, according to one embodiment, an absorbent layer 72 forthe wavelength of the laser beam 66 whereon a film 74 of at least onebio-ink is affixed. In the following part of the description, film meansthat the bio-ink occupies a volume with a thickness (the dimension in adirection perpendicular to the plane of impact Pi) of less than 500 μm.Unlike a tank, the fact that bio-ink is packaged as a film can make itpossible to avoid settling phenomena.

The absorbent layer 72 is made of a material adapted to the wavelengthof the laser beam 66 to transform the light energy into a punctualheating of the absorbent layer 72. The donor substrate 70 is preferablyso positioned that the optical system focuses the laser beam at theabsorbent layer 72. According to one embodiment, the absorbent layer 72is made of gold, titanium, or another component according to thewavelength of the laser beam 66. According to another embodiment, thedonor substrate 70 includes no absorbent layer 72. In this case, thelaser beam 66 energy is absorbed by the ink.

The donor substrate 70 preferably comprises a blade 76 made of amaterial transparent for the wavelength of the laser beam 66 whichincludes, on one of its faces, a coating corresponding to the absorbentlayer 72. The presence of the blade 76 imparts stiffness to the donorsubstrate 70 which makes it possible to handle and keep the ink and/orthe absorbent layer 72 substantially flat in the impact plane Pi. Thebio-ink film 74 comprises a free surface 78 which is spaced from theabsorbent layer 72 by a distance E corresponding to the thickness of thefilm 74 and which is spaced from the depositing surface 56 by a distanceL. The free surface 78 and the depositing surface 56 face each other. Asillustrated in FIG. 3, the laser beam 66 is adapted to produce a cavity80 at the interface between the absorbent layer and the bio-ink film 74which generates a droplet 82 which detaches from the free surface 78 tomove to the depositing surface 56.

In the following part of the description, a vertical direction isparallel to the gravitational force G and the up-down directioncorresponds to the direction of the gravitational force G. The directionof the laser beam 66 and the direction of the droplet movement areparallel to the vertical direction.

Upward Printing:

According to one characteristic of the invention, the laser beam 66 andthus the movement of the droplet 82 are oriented in the oppositedirection relative to the gravitational force G. The free surface 78 ofthe bio-ink film 74 is thus directed upwards. When moving from thebio-ink film 74 to the depositing surface 56, the droplet 82 movesupwards in the down-up direction.

This configuration provides the following advantages:

-   -   It limits the occurrence of settling phenomena, with the bio-ink        being in the form of a film,    -   It makes it possible to obtain a substantially constant        thickness E for the bio-ink film 74, with the influence of the        gravitational force G on the shape of the free surface 78 of the        film 74 being limited by the free surface 78 being oriented        upward,    -   It makes it possible to use a wide range of bio-ink when an        independent absorbent layer 72 of the bio-ink film 74 is used to        transform the light energy into a punctual heating.        Almost Aero Kinetic Energy at the Time of Depositing a Droplet        onto the Receiving Substrate:

The formation of a droplet 82 from a bio-ink film will depend on manyparameters, mainly the characteristics of the bio-ink, thecharacteristics of the laser beam and the conditions of implementation.FIGS. 4A to 4D show the evolution during the time of deformation of thefree surface of the bio-ink film, which results, or not, in theformation of a droplet, for different values of the beam laser 66energy, with the latter having an energy of 21 μJ in FIG. 4A, 35 μJ inFIG. 4B, 40 μJ in FIG. 4C and 43 μJ in FIG. 4D.

For the same bio-ink and under the same embodiment conditions, it can benoted that several rates exist, according to the energy of the laserbeam. As illustrated in FIG. 4A, if the energy of the laser beam is lessthan a lower threshold, the droplet does not detach from the bio-inkfilm 74. Since the maximum height of the deformation 84 generated at thefree surface 78 of the ink film 74 is less than the distance L betweenthe film 74 and the depositing surface 56, no element is printed.According to the selected example, the lower threshold ranges from 21 μJto 35 μJ. As illustrated in FIG. 4D, if the energy of the laser beam isabove an upper threshold, the gas bubble 80 produced inside the filmbreaks at the free surface thus causing the uncontrolled projection ofmicrodroplets. According to the selected example, the upper thresholdranges from 40 μJ to 43 μJ.

Between the lower and upper thresholds, as illustrated in FIGS. 4B and4C, the rate is such that it enables the formation of a jet. If thedistance L separating the film 74 and the depositing surface 56 issufficient, this rate enables the formation of a droplet. Preferably,the distance L is of the order of 1 to 2 mm to enable the formation of adroplet and not of a continuous jet which stretches out from the film upto the depositing surface. This configuration limits the risks ofcontamination of the biological tissue achieved by the bio-ink.

According to another characteristic of the invention, for the samebio-ink and under the same embodiment conditions, the distance Lseparating the ink film 74 and the depositing surface 56 and/or the beamlaser 66 energy are so adjusted that the kinetic energy of the dropletis almost equal to zero when the droplet 82 contacts the depositingsurface 56, as illustrated in FIG. 5D. This configuration limits therisk of damaging the elements to be printed which are cells. Almostequal to zero means that the kinetic energy is null or slightly positiveto enable the droplet to settle down on the depositing surface 56. Thiscircumstance is made possible because the droplet 82 moves in theopposite direction relative to the gravitational force G.

The distance L separating the bio-ink film 74 and the depositing surface56 is preferably stationary. Thus, the laser beam 66 energy is soadjusted that the kinetic energy of the droplet is almost equal to zerowhen the droplet 82 contacts the depositing surface 56. Whatever theapplication, printing with a rate which leads to a deposit at zero speedreduces the risks of the droplet splashing when contacting thedepositing surface.

Calibration Technique:

As indicated above, the formation of the droplet is not related to theenergy of the laser beam only. It is also related to the nature of thebio-ink especially the viscosity, the surface tension and the conditionsof implementation thereof.

FIGS. 5A to 5D, 7A to 7D illustrate a calibration method for determiningthe energy of the laser beam for obtaining an optimal rate for theformation and the deposition of droplets, specifically a rate that leadsto a deposit at zero speed at a given distance L. FIGS. 5A to 5D showsome of the steps of forming a droplet 82 between the time of impact ofthe laser beam shown in FIG. 5A and the deposition of the droplet 82 onthe depositing surface 56. According to one characteristic of theinvention, the calibration method for adjusting the laser energycomprises the steps of measuring an included angle Θ of a deformation 86of the free surface 78 of the film 74 of the bio-ink at a set time T1after the impact of the laser beam 66 and of adjusting the energy of thelaser beam 66 according to the measured value of the included angle Θ.

As illustrated in FIGS. 5B, 7A and 7B, the deformation 86 has asymmetrical shape with respect to a median axis Am parallel to thevertical direction. This deformation 86 includes a vertex S centered onthe median axis Am. This vertex S corresponds to the deformation area 86furthest from the rest of the free surface 78 of the film 74.

In a plane containing the median axis Am, the vertex S is extended by afirst flank 88 on one side of the median axis Am and by a second flank88′ on the other side of the median axis Am, with both flanks 88, 88′being symmetrical with respect to the median axis Am. Each face 88, 88′comprises a point of inflexion. The first flank 88 comprises a firsttangent Tg1 at its inflexion point and the second flank 88 includes asecond tangent Tg2 at its inflexion point, with the two tangents Tg1 andTg2 being secant at a point of the median axis Am.

The included angle Θ corresponds to the angle formed by the tangents Tg1and Tg2 and facing the film 74 (or downwards). For the formation of adroplet, the included angle Θ must be smaller than or equal to a firstthreshold Θ1. As illustrated in FIG. 7A, if the included angle Θ isabove the first threshold Θ1, the energy of the laser beam is thus notsufficient to generate a droplet. On the contrary, as shown in FIG. 7B,if the included angle Θ is smaller than the first threshold Θ1, thelaser beam energy is sufficient to generate a droplet.

To obtain a near-zero kinetic energy when the formed droplet reaches thedepositing surface 56 at a distance L from the free surface 78 of thefilm 74, the included angle Θ must be greater than or equal to a secondthreshold Θ2. The value of the included angle Θ is preferably determinedusing a take at the time T1 of the deformation 86. In one embodiment,the take is performed using a camera, the axis of sight of which isperpendicular to the vertical direction.

The time T1 advantageously depends on the film thickness and very littlevaries from one ink to another. The time T1 is advantageously of theorder of 4 to 5 μs from the impact of the laser beam for a thickness Eof the film of the order of 40 to 50 μm. This time T1 is illustrated inFIG. 5B.

The first threshold Θ1 is approximately equal to 105°. Thus, if at thetime T1 the included angle Θ is less than or equal to 105°, the laserbeam energy is sufficient to generate a droplet 82. The second threshold02 depends on the distance L between the depositing surface 56 and thefree surface 78 of the ink film 74. The second threshold Θ2 is inverselyproportional to the distance L.

The second threshold Θ2 is high and equal to approximately 80° for asmall distance L, of the order of 1 mm. A relatively small distance Lwill be preferred to reduce the stresses in the jet and at the time ofthe contact of the droplets with the depositing surface. The secondthreshold Θ2 is low and equal to approximately 50° for a substantialdistance L of the order of 10 mm. A relatively long distance L will bepreferred if a remote printing is desired, for instance, if the donorsubstrate 70 has larger dimensions than those of the well at the bottomof which the depositing surface 56 is positioned. This technique forcalibrating the laser beam energy makes it possible to optimize thespeed of the jet by reducing it in order to limit the risks of damagingthe elements contained in the ink particularly at the time of depositionon the depositing surface 56.

Thickness of the Ink Film:

The bio-ink composition preferably includes a high concentration inelements to be printed 62 in order to obtain a biological tissue with ahigh concentration in cells. In this case, as illustrated in FIG. 3, thedroplet 82 includes a volume fraction with a high concentration inelements to be printed 62.

For bio-inks with a high concentration, the thickness E of the film 74is of the order of 40 to 60 μm. In order to improve the accuracy of thedeposition of the elements to be printed, the film 74 of bio-inkadvantageously has a thickness E ranging from 1.5D to 2D, with D beingthe diameter of the elements to be printed 62 which have anapproximately spherical shape or the diameter of a sphere wherein anelement to be printed 62 is inscribed. According to one embodiment, thebio-ink film 74 has a thickness E greater than or equal to 20 μm forsmaller elements to be printed which have a diameter of the order of 10to 15 μm. The thickness E of the film may be of the order of 400 μm whenthe elements to be printed 62 are aggregates of cells. Generally, thethickness E of the film is less than 100 μm when the elements to beprinted 62 are unit cells.

Preferably, the film 74 is characterized by a (dimension of the freesurface 78)/film thickness 74) ratio greater than or equal to 10, andadvantageously greater than or equal to 20. The size of the free surface78 corresponds to the largest dimension of the free surface 78 of thefilm 74 in a plane parallel to the plane of impact Pi.

Printing Technique Combining a Laser-Type Print Head and a Nozzle PrintHead:

According to another characteristic of the invention, the printingmethod uses at least one laser-type print head for at least a firstbio-ink and at least one nozzle print head for at least a secondbio-ink. This combination makes it possible to increase the productionrate. Nozzle print head means a print head which comprises an orificethrough which the second bio-ink passes. Thus, a nozzle print head maybe a print head of the ink-jet type, a microvalves print head, a printhead of the bio-extrusion type.

Each print head of the laser type is preferably the same as the onedescribed in FIG. 3. However, the invention is not limited to this typeof laser print head. Using laser-type print heads as described in FIGS.1 and 2 or other print heads can be considered. The nozzle print headsare not described any further since they are preferably identical withthose of the prior art. In the case of a biological tissue comprisingdisjoint cells separated by extracellular materials, the extracellularmaterials are preferably deposited by the nozzle print head(s) and thecells are preferably deposited by the laser-type print head(s).

As the extracellular materials are less sensitive to shear effects, theycan be deposited using a nozzle print head. As the bio-ink cartridgesintended for the nozzle print heads have a very much higher volume thanthe volume of ink (of the order of 40 μm) supported by a donor substrate70 intended for a laser-type print head, the materials of theextracellular matrix can be deposited with a high flow rate. Although anozzle print head is capable of depositing inks with a high flow rate,as each donor substrate intended for a laser type print head supports avery small volume of ink, it is necessary to change these frequently,which tends to increase the removal time relative to a nozzle printhead.

According to another characteristic, the laser type head(s) or printingand the nozzle print head(s) are integrated in the same machine and movein the same coordinate system. This configuration makes it possible tosimplify the relative positioning of the various print heads, to improvethe accuracy of the removal and to guarantee the integrity of theprinted elements.

Printing Device Comprising a Donor Substrates Storage Chamber:

FIGS. 8 to 11 show a printing device according to one embodiment of theinvention. The printing device 100 includes a chassis supporting a printhead 102 of the laser type and several print heads 104, 104′, 104″ ofthe ink-jet type. The chassis 100 includes a coordinate system X, Y, Z,with the Z axis being oriented in the vertical direction, and with theX, Y plane corresponding to a horizontal plane.

The print heads 102, 104, 104′, 104″ are stationary with respect to thechassis 100 and so positioned that the droplets are emitted verticallyupwards. The print heads 102, 104, 104′, 104″ are offset in a firstdirection parallel to the Y axis. In one embodiment, the print heads104, 104′, 104″ of the ink-jet type are joined together. The print head102 of the laser type is spaced from the print heads 104, 104′, 104″ ofthe ink-jet type.

The printing device also comprises a mobile chassis 106, a system forguiding and moving the mobile chassis 106 relative to the chassis 100 inthree directions parallel to the X, Y, Z axes and a control system forcontrolling the displacements of the mobile chassis 106. The guide anddisplacement system and the control system are so selected as to achievea micrometric precision as regards the movements of the mobile chassis106 relative to the chassis.

As illustrated in FIG. 10, the mobile chassis 106 includes a frame 108for releasably attaching at least one receiving substrate 58. When it issecured to the mobile chassis, the movements of the receiving substrate58 are controlled with micrometric precision. The print head 102 of thelaser type comprises a hollow cylindrical body 110, stationary relativeto the chassis, which contains a part of an optical system and whereon atubular portion 112 is positioned which includes an upper end 114 whichopens into a horizontal plane. These elements are so configured that alaser beam guided by the optical system scans the section of the upperend 114.

Each donor substrate 70 has the shape of a disk positioned on a base116. According to one embodiment illustrated in FIG. 11, each base 116has the shape of a tube which comprises, at its upper edge, a recess 118which has a diameter identical to that of a donor substrate 70 and aheight sufficient to hold same. This recess 118 thus makes it possibleto position a donor substrate 70 relative to the base which supportssame.

The upper end 114 and the base 116 have shapes that cooperate with eachother so that the base 116 is immobilized in a given position relativeto the upper end 114 and thus with respect to the X, Y, Z system of thechassis. According to one embodiment, the base 116 includes an outerflange 120 which bears against the upper end 114 and makes it possibleto position the base along the Z axis. Under the flange 120, the base116 includes a frustoconical surface 122 which cooperates with a taperedportion provided inside the tubular portion 112. These shapes make itpossible to center the base 116 relative to the tubular portion 112 andto position same in an XY plane. Magnetic materials can preferably beused to improve the positioning of the base 116 relative to the tubularportion 112.

The printing device 124 advantageously comprises a chamber so configuredas to store at least one base 116. The chamber 124 comprises at leastone opening 125 for inputting and outputting the stored bases 116.According to one embodiment, the chamber 124 has a parallelepiped shape.The chamber 124 preferably has dimensions adapted to store severalbases. The printing device can thus successively print several bio-inkswith the same print head 102 of the laser type.

The bases 116 are stored on a base plate 126 which comprises recesses128, i.e. one recess for each base 116. The base plate 126 has anelongated shape and comprises, on its whole length, U-shaped notches128. According to a first alternative solution illustrated in FIG. 9,the length of the base plate 126 is oriented along the Y axis.

According to a second preferred embodiment, the length of the base plate126 is oriented along the X axis and the notches 128 are open towardsthe print heads. The chamber 124 advantageously includes, on a firstside facing the print heads, a first opening 125 enabling the bases 116to come out and, on another side, a second opening 125′ for introducingthe bases 116.

According to one embodiment, the chamber 124 includes a guide system forpositioning the base plate 126, for example a rail, with the base plate126 comprising, in the lower part, a groove the cross-section of whichengages with that of the rail. This rail opens at the second opening125′. It is preferably oriented along the X axis.

The chamber 124 comprises containment means for preserving, inside thechamber, an atmosphere adapted to bio-inks, in particular as regardstemperature and/or hygrometry. Such containment means are provided inparticular at each opening 125, 125′. They may take the form of abarrier or an air curtain.

In addition to the chamber, the printing device includes a mobile clamp130 to move the bases between the chamber 124 and the print head 102 ofthe laser type. In a first alternative solution, the mobile clamp 130 issecured to a mobile carriage 132, independent of the mobile chassis 106,which is configured to move in the X, Y, Z directions. According toanother alternative solution, the mobile clamp 130 is secured to themobile chassis 106.

According to one embodiment, the printing device includes a shootingdevice (not shown) the line of sight of which is perpendicular to thevertical direction and faces the upper surface of the donor substrate.This device can be used to calibrate the energy of the laser beam of theprint head 102 of the laser type.

Method for Producing a Biological Tissue using Bio-printing:

The first step of said method consists in generating a three-dimensionaldigital representation of the biological tissue to be printed. In FIG.12, a portion of such a representation is shown (140) as a cube whichhas a first volume region 142 positioned inside within a second volumeregion 144 itself positioned in a third volume region 146. For thepurpose of the description, the representation 140 is greatlysimplified.

Each volume region 142, 144, 146 is colored or textured differently,with each color or texture corresponding to a set of characteristicsamong the following (not limited) ones: material, manufacturing means,trajectory, . . . Each color or texture preferably corresponds to abio-ink. All the volume regions 142, 144 and 146 are closed.

The representation advantageously comprises a plurality of smallelementary volumes which have different colors or textures depending onthe volume region which they belong to. According to one embodiment, therepresentation 140 originates from a computer file of the PLY type.

The second step of the method consists in slicing the representation 140into a succession of stacked layers along an axis Z. In FIG. 13, a layer148 of the 140 representation has been separated. When slicing therepresentation 140, in line with a change of volume region, each layercomprises an edge which corresponds to a new region.

As illustrated in FIG. 13, the layer 148 comprises a first region 142′which corresponds to the first volume region 142, a second region 144′which corresponds to the second volume region 144 and a third region146′ which corresponds to the third volume region 146. For each layer,the regions 142′, 144′, 146′ are colored or textured according to thecolor or texture of the volume regions 142, 144, 146.

Each layer has a thickness c which is determined according to the heightof the printed droplets. If the layer comprises only one material to beprinted, the layer has a thickness substantially equal to the height ofa droplet. When the layer comprises several materials to be printed, ina first alternative solution, the layer has a thickness equal to thelowest common multiple of the heights of the droplets associated witheach material. This alternative solution has the advantage of minimizingany shifting on the whole height of the object to be printed and toachieve fast printing.

According to a second alternative solution, the layer has a thicknessequal to the highest common factor of the heights of the dropletsassociated with each material. This alternative solution has theadvantage of increasing the resolution and the number of layers.

For example, if the first material is printed by laser bio-printing, theprinted droplets have a height of the order of 10 μm. If the secondmaterial is printed using micro-valves bio-printing, the printeddroplets have a height of the order of 100 μm. In the first alternativesolution, the layers have a thickness of the order of 100 μm. In thesecond alternative solution, the layers have a thickness of the order of10 μm. Preferably, each layer comprises a plurality of small elementary,for example triangular, polygons which have different colors accordingto the region which they belong to. The object to be printed thuscorresponds to a set of layers which each comprise a set of polygons,each having an associated color or texture.

A third step of the method consists in determining, for each layer, theposition of the droplets to be printed of each bio-ink according to thecolored or textured regions 142′, 144′, 146′ and the expected volume ofeach droplet. For this purpose, each region 142′, 144′, 146′ of eachlayer is filled with ellipses 142″, 144″, 146″, the dimensions of whichdepend on the dimensions of the droplets of the bio-ink to be printed insaid region, as shown in FIG. 14.

For each region, the ellipses have the same dimensions. All the ellipseshave parallel focal axes. The elliptical shape makes it possible toadjust the distances between the droplets in two directions (a firstdirection parallel to the focal axes and a second directionperpendicular to the first one). The center of each ellipse correspondsto the position of the center of a droplet. The ellipses are positionedregion by region, in the descending order of sizes, thus the largerellipses arranged in the region 146′ are positioned first and thesmaller ellipses arranged in the region 142′ are positioned last.

Preferably at a region change, the positioning is optimized according totwo criteria:

-   -   the maximum ratio of elementary polygons having the right color        or texture in an ellipse, of the order of 75% for example,    -   the minimum ratio of elementary polygons having the wrong color        or texture in an ellipse, of the order of 5% for example.        Ellipses overlapping can be tolerated.

A fourth step of the method consists in synchronizing the movement ofthe depositing surface 56 whereon the bio-ink droplets are printed andthe various print heads. For bio-laser printing, the laser focusing areais the center of each laser printed ellipse, and each ellipse is thesubject of a laser pulse. In this case, the depositing surface isstationary, the laser scans the entire depositing surface. For adepositing surface larger than the donor substrate, the substrate canalso be moved (where the depositing surfaces are tipped on) insynchronization with the laser scan.

For a nozzle bio-printing, the center of each ellipse corresponds to theassumed point of impact of a droplet on the depositing surface 56. Inthis case, the print nozzle is stationary, the substrate moves. However,the print nozzle may be movable.

Applications:

Bio-printing according to the invention can be used to produce:

Implantable tissues for regenerative medicine,

Individualized tissues, made from the patient's cells, making itpossible to select the treatments, in vitro, and to develop customizedtherapeutic solutions,

Predictive models reproducing the physiology of sound human tissues ortissues affected by a pathology in order to predictively test theefficacy or the toxicity of the molecules, of ingredients and of thedrug candidates.

By way of example and without limitation, the biological tissue is abone tissue.

Although described as applied to bio-inks, the invention is not limitedto this application. The method and the device according to theinvention can thus be used to print any liquid ink, as a solution or asuspension. Other suitable inks include but are not limited to the inksused in electronic coatings, in materials or in watchmaking.

For example and without limitation, the inks can be composed of:

-   -   Precious (specifically gold, silver, platinum, rhodium and        palladium) or semi-precious (titanium, zirconium, copper)        metals,    -   Functional alloys,    -   Organic materials,    -   Sol-gel systems,    -   Ceramics or microcomposites or nanocomposites.

These different materials make it possible to produce various types ofcoatings:

-   -   Anticorrosive coatings,    -   Coatings with a high chemical resistance,    -   Bio-functional (antibacterial, antimicrobial, biocompatible)        coatings,    -   Coatings for food contact,    -   Coatings modifying the surface energy,    -   Release coatings,    -   Electro-technical (insulating, antistatic or conductive)        coatings    -   Wear resistant coatings    -   Coatings modifying the optical properties (anti-reflective,        photocatalytic, IR/UV barrier),    -   Coatings affecting the haptic sense    -   Coatings for reducing the friction coefficient,    -   Coatings making it possible to increase durability at high        temperatures.

The invention claimed is:
 1. A method for printing with at least oneink, with the method comprising: focusing a laser beam so as to generatea cavity in an ink; forming at least one ink droplet from a free surfaceof the ink film; depositing the droplet onto a depositing surface of areceiving substrate positioned at a given distance from the film; andorienting the laser beam in a direction opposite a gravitational force,with the free surface of the film being oriented upwards towards thedepositing surface placed above the ink film; wherein the distanceseparating the ink film and the depositing surface and/or the beam laserenergy are so adjusted that kinetic energy of the droplet is almostequal to zero when the droplet contacts the depositing surface.
 2. Aprinting method according to claim 1, wherein the film has a thicknessof less than 500 μm.
 3. A printing method according to claim 1 wherein:the film has a dimension of the free surface of the film on the filmthickness ratio greater than or equal to
 10. 4. A printing methodaccording to claim 1, wherein the distance separating the ink film andthe depositing surface is equal to 1 to 2 mm.
 5. A printing methodaccording to claim 1, further comprising a preliminary phase ofcalibration of the energy of the laser beam which comprises measuring anincluded angle of deformation of the free surface of the ink film at aset time after an impact of the laser beam, and adjusting the laser beamenergy according to the measured value of the included angle.
 6. Aprinting method according to claim 5, wherein the laser beam energy isso adjusted that the included angle is less than or equal to 105°.
 7. Aprinting method according to claim 5, wherein the laser beam energy isso adjusted that the included angle is greater than or equal to a secondthreshold to obtain the kinetic energy almost equal to zero when theformed droplet reaches the depositing surface.
 8. A printing methodaccording to claim 7, wherein the second threshold depends on thedistance between the depositing surface and the free surface of the inkfilm.
 9. A printing method according to claim 8, wherein the secondthreshold is equal to approximately 80° for the distance of an order of1 mm.
 10. A printing method according to claim 5, wherein the includedangle measuring time is of an order of 4 to 5 μs as from the impact ofthe laser beam.
 11. A printing method according to claim 1, wherein theink film has a thickness greater than 20 μm.
 12. A printing methodaccording to claim 1, wherein the ink film has a thickness ranging from40 to 60 μm for bio-inks with a high concentration of elements to beprinted.
 13. A printing method according to claim 1, wherein for abio-ink with a low concentration of elements to be printed, the bio-inkfilm has a thickness ranging from 1.5D to 2D, with D being the diameterof the elements to be printed which have an approximately sphericalshape, wherein the diameter of a sphere in which an element to beprinted is inscribed.